Secreted alkaline phosphatase: an internal standard for expression of injected mRNAs in the Xenopus oocyte.
The Xenopus oocyte is widely used to study the various aspects of eukaryotic cell structure and function. It is also being used increasingly in expression cloning of cDNAs encoding proteins for which there are no structural data. One of the drawbacks of the Xenopus oocyte system is that individual oocytes taken at the same time from the same frog vary considerably in the amount of protein synthesized from the same amount of injected mRNA. In this report we describe the preparation and use of the mRNA for a secreted mutant form of human placental alkaline phosphatase as an internal, coinjected standard to monitor translation in oocytes. Secreted alkaline phosphatase can be readily determined in the medium of cultured oocytes by using a standard colorimetric assay. The amounts of alkaline phosphatase secreted into the medium were shown to parallel the level of expression of two membrane proteins. This permits rapid identification and selection of those oocytes that efficiently express injected mRNAs. The procedure yields more precise data and results in an enormous saving of time and expense, especially in investigations that involve complex measurements on individual oocytes.
Tate
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Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Urade
R
R
Micanovic
R
R
Gerber
L
L
Udenfriend
S
S
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D005260:Female; D015870:Gene Expression; D005819:Genetic Markers; D006801:Humans; D007668:Kidney / Q000032:analysis; D009154:Mutation; D009865:Oocytes / Q000201:enzymology*; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D014982:Xenopus laevis
D005819:Genetic Markers; D012333:RNA, Messenger; D000469:Alkaline Phosphatase